Antibody against cleavage product of vimentin

ABSTRACT

The present invention provides the antibody that react with the cleavage product of vimentin, but not with intact vimentin.

FIELD OF THE INVENTION

[0001] The present invention relates to an antibody against the cleavageproduct of vimentin; a method for detecting apoptosis using thisantibody; and a use for this antibody.

BACKGROUND OF THE INVENTION

[0002] Apoptosis is cell death in multicellular organisms. Surplus cellsgenerated during developmental processes, cells no longer needed in anadult, cells damaged by radiation or chemical substances, or dangerouscells such as tumor cells are led to cell death by apoptosis, thusremoved from a body.

[0003] Caspases are proteolytic enzymes (protease) that play a key roleduring apoptosis. Research on apoptosis has rapidly expanded since the1990s. One of the key factors to have promoted the research is theidentification of a caspase family of proteases that involves executionof apoptosis (Thornberry, N. & Lazebnik, Y. (1998) Science, 281,1312-1316). At least 10 or more members of the caspase family areidentified in mammals. Caspases are also shown to be present as aninactive precursor in normal cells. When apoptosis is initiated for acell to die, an initiator caspase in the caspase family activates itselfby limited proteolysis (processing). The activated initiator caspaseactivates another caspase by partially cleaving it, the cleaved caspaseactivates another caspase, and the process continues one after another.This amplification cascade mechanism is thought to achieve the wholeactivation. All caspases cleave the C-terminal side of a specificaspartic acid residue in protein, but the cleavage efficiency of eachmember of the caspase family varies depending on amino acid sequencesnear the cleavage site.

[0004] Apoptosis triggered by the stimulation of the anti-Fas antibodyis the best analyzed apoptosis and thought to play a central role amongadults (Nagata, S. (1997) Cell 88, 355-365). Caspase-8, involvingexecution of apoptosis is first activated among the caspase family incells after stimulation with the anti-Fas antibody, and functions as aninitiator (Boldin, M. P., Goncharov, T. M., Goltsev, Y. V. & Wallach, D.(1996) Cell 85, 803-815; Muzio, M., Chinnaiyan, A. M., Kishkel, F. C.,O'Rourke, K., Shevchenko, A., Ni, J., Scaffidi, C., Bretz, J. D., Zhang,M., Gentz, R., Mann, M., Krammer, P. H., Peter, M. E. & Dixit, V. M.(1996) Cell, 85, 817-827).

[0005] Several methods for detecting activation of a caspase have beenemployed, such as 1) detecting processing of a caspase or activationusing an antibody recognizing a caspase; or 2) measuring proteaseactivity using a substrate analog. Any of these methods, however, have adrawback in that the ability to distinguish between members of caspasefamily is limited. In the method of 1), production of a specificantibody capable of recognizing both an inactive precursor and an activetype is often difficult.

SUMMARY OF THE INVENTION

[0006] It is the objective of the present invention to provide anantibody against the cleavage product of vimentin that is a maincomponent of an intracellular skeletal protein; a method for detectingapoptosis using said antibody; and the use of said antibody.

[0007] As a result of intensive and extensive research toward theabove-mentioned objective, the inventors have finally found thatvimentin in an apoptotic cell is specifically cleaved by caspase-8, andthey have succeeded in producing an antibody capable of detecting thespecific cleavage of vimentin.

[0008] That is, the present invention relates to an antibody that reactswith the cleavage product of vimentin but does not react with intactvimentin. The cleavage product of said vimentin is the one cleaved byaction of caspase (e.g., caspase-8). The abovementioned antibody can beeither a polyclonal antibody or a monoclonal antibody.

[0009] Further, the present invention relates to a method for detectingcaspase activity or a method for detecting apoptosis, comprisingallowing the above antibody to react with cleavage product of vimentinand detecting the resulting reaction product.

[0010] Furthermore, the present invention relates to a reagent fordetecting apoptosis containing the above antibody.

[0011] The present invention will now be described in detail. Thisspecification includes part or all of the contents as disclosed in thespecification and/or drawings of Japanese patent Application No.11-193235, which is a priority document of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0012]FIG. 1 shows a schematic diagram of the cleavage site by caspasein vimentin.

[0013]FIGS. 2 and 3 show photographs of Western blot analysis.

[0014]FIG. 4 shows photographs of indirect immunofluorescence stainingof apoptotic cells.

DETAILED DESCRIPTION OF THE INVENTION

[0015] The present invention relates to an antibody specificallyrecognizing cleavage product of vimentin but not reacting with the wholevimentin (intact vimentin), a substrate of a caspase (e.g., caspase-8)activated at the initial stage of apoptosis. To detect the activity ofcaspases, the present inventors have produced an antibody reacting witha cleavage site of a substrate cleaved by a caspase by focusing onchanges in the substrate (vimentin) not on the caspase itself.Therefore, activation of a caspase can now be detected with highsensitivity regardless of sample scale, such as tissue and cell.

[0016] Among amino acid sequences of vimentin protein, the presentinventors have identified an aspartic acid residue at a specific sitewhich is cleaved by caspase-8. Oligopeptides having a sequence onN-terminal side or C-terminal side of the aspartic acid residue arechemically synthesized and used for immunizing a rabbit. After severaltimes of immunization, anti-serum is obtained. Then an antibody thatbinds strongly to the oligopeptide used for immunization in anti-serumis purified. The purified antibody does not bind to an intact vimentinprotein, but specifically bind to a product cleaved by caspase-8. Byusing this antibody in the Western blot analysis and immunostaining ofcells and tissues, the detection of activation of caspse-8 becomespossible through cleavage of vimentin either in vitro, in tissue sampleor in cell sample.

[0017] The term “antibody” in the present invention represents the wholeantibody molecule or its fragments (e.g., Fab or F(ab′)₂ fragment),which can bind to the cleavage product of vimentin as an antigen. Thisantibody of this invention may be a polyclonal or monoclonal antibody.

[0018] Antibodies of the present invention can be produced by variousmethods. Such production methods of antibodies are known in the art.(For example, see Harlow E. & Lane D., Antibody, Cold Spring HarborLaboratory press (1988))

[0019] Preparation of Antibodies Reacting with Cleavage Products ofVimentin

[0020] (1) Preparation of Antigens

[0021] Vimentin on which caspases act is an intermediate filamentprotein, characteristics in mesenchymal cells, such as fibroblasts andleukocytes.

[0022]FIG. 1 is a scheme showing a site of an intermediate filamentprotein, vimentin, to be cleaved by a caspase when apoptosis isinitiated by treatment with anti-Fas antibody. Caspases used in thepresent invention include caspase-3, caspase-6 or caspase-8, preferably,caspase-8. For example, caspase-8 recognizes and cleaves C terminal sideof Asp-259 of an amino acid sequence of vimentin in a human or mouse(SEQ ID NO: 3 for a human, SEQ ID NO:4 for a mouse). Caspase-3recognizes and cleaves C-terminal side of Asp-85; caspase-6 recognizesand cleaves C terminal side of Asp-429. According to the presentinvention, the two types of antibody can be produced against vimentinprotein or peptide which is cleaved by caspase-8 between Asp-259 andVal-260: an antibody recognizing the resulting N-terminal fragment(referred to as V1 antibody) and an antibody recognizing the resultingC-terminal fragment (referred to as V2 antibody). See FIG. 1.

[0023] When the V1 antibody is produced, a protein or peptide fragmentthat has the maximum of 259 amino acid residues, preferably 6 amino acidresidues, or more preferably 5 amino acid residues, from Asp-259 to the1^(st) Met on the N-terminus among an amino acid sequence shown in SEQID NO:3 or 4 can be used as an antigen. Similarly, when the V2 antibodyis produced, a protein or peptide fragment that has the maximum of 207amino acid residues, preferably 6 amino acid residues, more preferably 5amino acid residues, from Val-260 to Glu-466 on the C-terminus among anamino acid sequence shown in SEQ ID NO: 3 or 4 can be used as anantigen. Further to improve the antigenicity, each of the above proteinor peptide fragment is preferably allowed to perform coupling reactionat the terminus other than the one resulted from the cleavage by acaspase. It is preferable for facilitating the coupling reaction that aCys residue is bound to the protein or peptide terminal.

[0024] (2) Production of Monoclonal Antibodies against Cleavage Productof Vimentin

[0025] (i) Recovery of Antibody-Producing Cells

[0026] proteins or peptides as produced in (1) are administered asantigens to mammals, such as a rat, mouse, or rabbit. A dosage of theantigen per animal is 0.1 to 100 mg when no adjuvant is used, and 1 to100 μg when an adjuvant is used. The adjuvants include Freund's completeadjuvant (FCA), Freund's incomplete adjuvant (FIA), and aluminumhydroxide adjuvant. Immunization is principally carried out byintravenous, subcutaneous, or intraperitoneal injection. In additionintervals of immunization are not particularly limited. Immunization iscarried out for 1 to 10 times, preferably 2 to 5 times, at intervals offrom several days to several weeks, preferably 2 to 5 week-intervals.One to 60 days, preferably 1 to 14 days after the final immunization,antibody-producing cells are recovered. Examples of antibody-producingcells include spleen cells, lymph node cells, peripheral blood cells,preferably spleen cells or local lymph node cells.

[0027] (ii) Cell Fusion

[0028] The antibody-producing cells are allowed to fuse with myelomacells so as to obtain hybridomas. Myeloma cells to be fused with theantibody-producing cells may include generally available establishedcell lines of animals, such as a mouse. preferable established celllines used herein have drug selectivity and cannot survive in HATselection medium containing hypoxanthine, aminopterin, and thymidinewhen unfused, but can survive therein only when fused with theantibody-producing cells. Specific examples of myeloma cells includemouse myeloma cell lines, such as X63Ag.8.653, NSI/1-Ag4-1, and NS0/1;and rat myeloma cell lines, such as YB 2/0.

[0029] Next, the myeloma cells are fused with the antibody-producingcells. Briefly, 1×10⁶ to 1×10⁷ cells/ml of antibody-producing cells aremixed with 2×10⁵ to 2×10⁶ cells/ml of myeloma cells in an animal cellculture medium such as serum-free DMEM or RpMI-1640. Preferable cellratio of the antibody-producing cells to myeloma cells is 2:1 to 3:1.Then, fusion reaction is performed in the presence of a cell fusionpromoter. The cell fusion promotor is, for example polyethylene glycolof an average molecular weight 1,000 to 6,000 daltons. Further, acommercially available cell fusion device using electric pulsestimulation, e.g., electroporation can be used to fuseantibody-producing cells with myeloma cells.

[0030] (iii) Selection and Cloning of the Hybridomas

[0031] Hybridomas of interest are selected from the fused cells. First,cell suspension is appropriately diluted with such as RPMI-1640 mediumcontaining fetal calf serum, about 3×10⁵ cells/well are placed inmicrotiter plate wells, selection medium is added to each wells, and thecells are cultured while appropriately replacing selection medium. Cellsgrowing around 14 days after the start of cultures in selection mediumcan be obtained as hybridomas.

[0032] Next, supernatants from hybridoma cell cultures are examined byscreening for the presence of antibody reacting with cleavage product ofvimentin. Screening of hybridomas may be performed by any of theconventional methods and not particularly limited. For example, a partof the culture supernatant contained in a well in which a hybridoma hasgrown may be collected and subjected to screening using an enzymeimmunoassay, radioimmunoassay or the like.

[0033] Cloning of fused cells is performed by the limiting dilutionmethod or the like. Finally, hybridomas which are cells producingmonoclonal antibodies that react with cleavage product of vimentin butnot with intact vimentin are established.

[0034] (iv) Recovery of Monoclonal Antibodies

[0035] Monoclonal antibodies can be recovered from establishedhybridomas using conventional methods, such as a cell culture method,and ascites formation method.

[0036] In the cell culture method, hybridomas are grown in culture mediafor animal cells, such as 10% fetal calf serum-containing RPMI-1640 orMEM medium, or serum-free medium under conventional culture conditions(for example, at 37° C., under 5% CO₂) for 7 to 14 days. Then antibodiesare recovered from the culture supernatants.

[0037] In the ascites formation method, about 1×10⁷ cells of hybridomasare administered intraperitoneally to animals belonging to the samespecies of the mammals from which myeloma cells are derived so that alarge number of hybridomas are grown. After 1 to 2 weeks, ascitic fluidis collected.

[0038] When purification of antibodies is required in the abovedescribed recovery methods, antibodies can be purified by known methodssuch as an ammonium sulfate method, ion exchange chromatography, gelfiltration, and affinity chromatography. These methods may be usedindependently or in combination.

[0039] (3) Production of Polyclonal Antibodies Against the CleavageProduct of Vimentin

[0040] Antigens prepared as described above are administered to mammals,such as a rat, mouse, and rabbit. A dosage of antigens per animal is 0.1to 100 mg when no adjuvant is used, and 10 to 1,000 μg when adjuvant isused. The adjuvants used in this invention include Freund's completeadjuvant (FCA), Freund's incomplete adjuvant (FIA), and aluminumhydroxide adjuvant. Immunization is principally carried out byintravenous, subcutaneous, or intraperitoneal injection. Intervals ofimmunization are not particularly limited. Immunization is carried outfor 1 to 10 times, preferably 2 to 5 times, at intervals of from a fewdays to a few weeks, preferably 2 to 5 week-intervals. Six to 60 daysafter the final immunization, antibody titer is measured using ELISA(enzyme-linked immunosorbent assay), EIA (enzyme immunoassay) or RIA(radioimmuno assay). On a day that the maximum antibody titer is shown,blood is collected to obtain anti-serum.

[0041] Subsequently, reactivity of polyclonal antibodies in anti-serumagainst the cleavage product of vimentin is measured by ELISA, etc.polyclonal antibodies showing strong reactivity against the cleavageproduct of vimentin but showing no reactivity against intact vimentinare selected.

[0042] For example, polyclonal antibodies in anti-serum are applied toan affinity column to which the cleavage product of vimentin is fixed,thereby recovering antibodies (column adsorption fraction) reacting withthe cleavage product of vimentin. Then the resulting antibodies areapplied to an affinity column fixed with intact vimentin, therebyrecovering antibodies that do not adsorb but flow out are recovered.Finally obtained antibodies are subjected to ELISA to confirm whetherthey react with the cleavage product of vimentin but do not react withintact vimentin.

[0043] 2. Method for Detecting Apoptosis The above antibody may be usedto detect (e.g., quantify) the cleavage product of vimentin in thepresent invention. For example, a sample containing the cleavage productof vimentin may be incubated with the monoclonal antibody or polyclonalantibody of the present invention, followed by an anti-mouse IgGantibody labeled with an enzyme such as horseradish peroxidase (HRP).The amount of the cleavage product of vimentin in the sample may bedetermined by measuring the intensity of color developed during theenzymatic reaction using a measuring device. The measured value can beused as an indicator for detecting the activity of caspase. A greatervalue measured represents an increased activity of caspase, indicatingthat apoptosis of the test cell has progressed.

[0044] The antibody of the present invention may also be used to detectapoptosis by reacting the antibody with cells in a biological sample.For example, the sample to be assayed may be incubated with the aboveantibody. The cleavage product of vimentin present in the sample may bedetected and quantified by using an anti-mouse IgG antibody labeled withhorseradish peroxidase (HRP) etc. according to conventional methods.

[0045] A measured value greater than negative control indicates a higheractivity of caspase. On the contrary, a measured value less thanpositive control indicates a lower activity of caspase. These measuredvalues can be used as data for determining the progression of apoptosis.For example, they may be used as indicators of the progression ofautoimmune diseases such as systemic lupus erythematosus (SLE),autoimmune hemolytic anemia and Basedow's disease or acquiredimmunodeficiency syndrome (AIDS).

[0046] Reagent Containing the Antibody of the Present Invention

[0047] The antibody against the cleavage product of vimentin may be usedas various reagents in the present invention. For example, when theantibody is used as a reagent for detecting the cleavage product ofvimentin, the detection may be carried out using the procedure shown inSection 2 above. The reagent according to the present invention mayinclude, for example, an anti-mouse IgG antibody labeled with HRp, ananti-rabbit IgG antibody labeled with HRP, a substrate for HRp and abuffer, as well as the above antibody.

[0048] Alternatively, when the antibody of the present invention is usedas a reagent for immunohistochemical staining, the detection may becarried out according to a conventional procedure forimmunohistochemical staining. In this case, the reagent according to thepresent invention may include, for example, an anti-mouse IgG antibodylabeled with HRP, a fluorescently labeled anti-mouse IgG antibody, asubstrate for HRP and a buffer, as well as the above antibody. Forexample, various tissue microscopic sections obtained by biopsy of SLEpatient may be prepared by conventional methods to react with theantibody of the present invention. These sections may be incubated withan anti-mouse IgG antibody labeled with horseradish peroxidase (HRP) asa secondary antibody and then treated with a substrate3,3′-diaminobenzidine to develop brown. The brown area found in thesection indicates that caspase has become active in this area.

EXAMPLES

[0049] The present invention is further described in the followingexamples. These examples are provided for illustrative purposes only,and are not intended to limit the scope of the invention.

Example 1 Preparation of Antibodies

[0050] (a) Materials

[0051] peptides used for immunization were obtained from Biologica Co.(Nagoya, Japan). Activated hemocyanin (Inject Maleimide-KLH) wasobtained from pierce (Rockford,Ill, USA). FMP-activated cellulofine wasobtained from Seikagaku Corp. (Tokyo, Japan). Other biochemical reagentswere obtained from Sigma-Aldrich Japan (Tokyo, Japan).

[0052] (b) Coupling of Chemically Synthesized peptides to Carrierproteins

[0053] Synthesized peptides (2 mg) were dissolved in Dulbecco'sphosphate buffer (hereinafter referred to as PBS; Harlow E. & Lane, D.(1988) “Antibody” Cold Spring Harbor Laboratory press, Cold SpringHarbor, N.Y., USA). These peptides have the following sequences,respectively: Cys-Gln-Ille-Asp-Val-Asp (SEQ ID NO: 1; referred to assequence V1 and Val-Ser-Lys-pro-Asp-Cys (SEQ ID NO: 2; referred to assequence V2).

[0054] Sequences V1 and V2 correspond to five amino acid residues thatare immediately N- and C-terminal to the cleavage site by caspase-8 invimentin protein, respectively and each having Cys residue for couplingreaction at the terminus other than the cleavage site by caspase-8 (FIG.1). Sequences V1 and V2 were dissolved in 400 μl and 200 μl PBS,respectively. Each solution was mixed with 200 μl of activatedhemocyanin (Imject Maleimide-KLH) to start coupling reaction. Eachreaction mixture was rotated gently at room temperature for 2.5 hours.The reaction product was dialyzed against PBS at 4° C. to removeunreacted peptides. The resulting peptide-carrier conjugate was dilutedwith PBS to a final volume of 5 ml.

[0055] (c) Immunization

[0056] The above peptide-carrier conjugate was mixed well with Freund'sadjuvant (Harlow E. & Lane, D. (1988) “Antibody” Cold Spring HarborLaboratory press, Cold Spring Harbor, N.Y., USA) and subcutaneouslyinjected into a New Zealand white rabbit for immunization. Thepeptide-carrier conjugate containing 400 μg of the peptide was used perinjection. The rabbit was immunized for 4 times at days 1, 9, 22 and 31.The circulating blood was collected after 8 days of the finalimmunization to prepare antiserum by conventional methods.

[0057] (d) preparation of Affinity Column for Purifying SpecificAntibodies 0.3 g of an activated resin (FMP-activated cellulofine) wereswelled in distilled water (50 ml) and filled into Econo-Column(Bio-Rad, Hercules, Calif., USA). The resin in the column was furtherwashed with 10 ml distilled water. One milligram of the synthesizedpeptide (sequence V1 or V2) was dissolved in 10 ml coupling buffer (50mM Na₂CO₃/NaHCO₃, pH 8.5) and mixed with the resin in the column tostart coupling reaction. The coupling reaction continued overnight at 4°C. with rotation. The resin was further incubated with 20 ml blockingbuffer (50 MM Tris-HCL, pH 8.0, 0.1 M monoethanolamine) at roomtemperature for 4 hours with rotation in order to block unreacted resin.Subsequently, unreacted peptides were removed by washing sequentiallywith the following solutions (20 ml each): 1) distilled water; 2) 0.1MGly-HCl (pH 2.5); 3) distilled water; 4) washing buffer (20 MM Tris-HCl,pH 7.5, 1 M NaCl, 1% Triton X-100) and 5) distilled water.

[0058] The affinity column prepared was washed with 10 ml elution buffer(0.1 M Gly-HCl, pH 2.5), 10 ml distilled water, followed by 20 ml TBS(20 mM Tris-HCI, pH 7.5, 0.15 M NaCl) immediately before used foraffinity purification.

[0059] (e) Affinity purification of Specific Antibodies from Antisera

[0060] The antiserum (3 ml for sequence V1, 6 ml for sequence V2) wasloaded on the above column and mixed well with affinity resin therein.The column was allowed to stand at room temperature for 1 hour to causebinding the antibodies to the resin. To remove non-specifically boundproteins, the column was washed sequentially with the followingsolutions: 1) 10 ml TBS; 2) 30 ml washing buffer (supra); 3) 30 ml TBSand 4) 10 ml of 0.15 M NaCl. The specific antibodies were eluted with 4ml elution buffer (supra). The protein fraction eluted from the columnwas mixed immediately with 1 M Tris (0.2 ml) on ice to recover its pH toa neutral range.

[0061] Finally, 4 ml of anti-V1 antibody (2.75 mg/ml) and anti-V2antibody (0.15 mg/ml) were obtained, respectively. These purifiedantibodies were divided into aliquots and stored frozen at −20 ° C.

Example 2 Western Blot Analysis Using Specific Antibodies

[0062] Human T cell line Jurkat or SKW6.4 (4×10⁵ cells/ml) was inducedto undergo apoptosis by treatment with 200 ng/ml anti-Fas antibody(Medical & Biological Laboratories Co., Ltd., Nagoya, Japan). Thetreatment continued for 0 to 8 hours at the longest duration. The cells(2×10⁶ each) were collected by centrifugation (1,000 rpm/min, 5 min) andthen suspended in PBS. The cells were centrifuged again under the samecondition as defined above to remove culture medium in the sample. Eachcell sample was ultrasonically treated for 20 seconds in 40 μl samplebuffer (62.5 mM Tris-HCl, pH 6.8, 6 M Urea, 2% sodium dodecylsulfate(SDS), 10% glycerol, 0.003% bromophenol blue) for SDS-polyacrylamide gelelectrophoresis. Proteins in each cell lysate sample were separated on12% polyacrylamide gel electrophoresis (Laemmli, U.K. (1970) Nature,227, 680-685) and transferred onto a nitrocellulose membrane usingSemidry Blot transfer device (Nihon EIDO, Tokyo, Japan) to form Westernblots.

[0063] The Western blots were immunostained by enzyme chemiluminescencelabelling method (ECL) (Harlow E. & Lane, D. (1988) “Antibody” ColdSpring Harbor Laboratory press, Cold Spring Harbor, N.Y., USA). Aprimary antibody was a commercially available anti-vimentin antibody V9(8.8 μg/ml, Sigma-Aldrich Japan, Tokyo, Japan) or the anti-V1 or V2antibody (0.2 μg/ml). A secondary antibody was an anti-mouse or rabbitIgG antibody labeled with horseradish peroxidase (1000-fold dilution,Cappel, Durham, N.C., USA). Following incubation with the secondaryantibody, the Western blots were visualized using ECL plus kit (AmershamJapan, Tokyo, Japan). Immunostaining was carried out according to themanufacturer's instruction. The results are shown in FIGS. 2 and 3.

[0064]FIG. 2 shows Western blots of Jurkat cell lysate treated with theanti-Fas antibody. Following electrophoresis, the blots wereimmunostained with the anti-vimentin antibody (V9). Upon treatment withthe anti-Fas antibody for several hours, caspases cleaved vimentin (58kDa) at the maximum of three sites into fragments of different size.

[0065] When the lysate of cells undergoing apoptosis was subjected toWestern blot analysis using the anti-V1 or V2 antibody, these antibodiesspecifically stained only vimentin fragments generated by cleavage ofthe sequence Ile-Asp-Val-Asp (FIG.3). The anti-V1 antibody detected 30kDa and 19.5 kDa fragments, while the anti-V2 antibody detected 27 kDaand 21 kDa fragments because other caspases caused further fragmentationalmost simultaneously with or subsequently to the fragmentation bycaspase-8. On the other hand, neither the anti-V1 nor V2 antibodyreacted with intact vimentin (58 kDa) that did not undergo cleavage (seeFIG. 3, Lane of hour 0). Hence, FIG. 3 shows that the antibody of thepresent invention recognizes only vimentin fragments found in cellsundergoing apoptosis.

Example 3 Indirect Immunofluorescence Staining of Apoptotic Cells withSpecific Antibodies

[0066] Human T cell line Jurkat (4×10⁵ cells/ml) was induced to undergoapoptosis by treatment with 200 ng/ml anti-Fas antibody (Medical &Biological Laboratories Co., Ltd., Nagoya, Japan). Following thetreatment for 6 hours, the cells (1.5×10⁶) were collected bycentrifugation (1,000 rpm/min, 5 min) and then suspended in PBS. Thecells were centrifuged again under the same condition as defined aboveto remove culture medium in the sample.

[0067] The collected cells were fixed in cold methanol at −20° C. for 2min. The fixed cells were washed with PBS and subjected to indirectimmunofluorescence staining (Harlow E. & Lane, D. (1988) “Antibody” ColdSpring Harbor Laboratory press, Cold Spring Harbor, N.Y., USA). First,the cells were incubated with the anti-V1 or V2 antibody (0.2 μg/ml, inPBS containing 3% bovine serum albumin) at room temperature for 1 hourand washed with PBS to remove unbound antibodies. Next, a fluorescentlylabeled anti-rabbit IgG antibody (Alexa 448, Molecular probes, Eugene,Oregon, USA) was used as a secondary antibody, which was diluted500-fold and added to the above cells for incubation at room temperaturefor 30 min. The cells were washed with PBS to remove unbound secondaryantibodies. PBS had contained a DNA-staining reagent DAPI (0.1 μg/ml,Sigma-Aldrich) in order to visualize nuclear chromosomes. The cells werethen examined by Fluorescence Microscope IX70 (Olympus Optical Co.,Ltd., Tokyo, Japan).

[0068] The results are shown in FIG. 4. FIG. 4 shows a photograph of thecells are fluorescently stained with the anti-V2 antibody. The apoptoticcells could be identified by condensation and fragmentation of theirnucleus when visualized using DApI (see small arrows in FIG. 4). Theanti-V2 antibody was found to specifically stain only cells undergoingapoptosis. Cells that do not undergo apoptosis (see large arrows in FIG.4) are not stained with the anti-V2 antibody. As a control, Jurkat cellsuntreated with the anti-Fas antibody were similarly examined, indicatingthat none of these cells were stained with the anti-V2 antibody.

[0069] The antibody of the present invention was shown to react onlywith the cleavage product of vimentin, but not with intact vimentin.

[0070] The present invention can achieve the detection of the actualcleavage of substrates by caspase regardless of sample scale such asprotein, cell or tissue. Hence, the present invention enables one 1) toobserve the conversion of caspase-8 from its precursor into its activeform; 2) to find the active form of caspase-8 to actually function inplace within the cell; and 3) to selectively distinguish the cells inwhich caspase-8 has become active in cell populations.

[0071] The present invention provides the antibody that react with thecleavage product of vimentin, but not with intact vimentin. By using theantibody of the present invention, the initiator caspase that is inactive form and is cleaving proteins can be identified by detecting thecleavage product of vimentin.

[0072] Vimentin is highly expressed in various developing tissues. Also,increased expression of vimentin is often observed when the tissuebecomes cancerous. Accordingly, the antibody of the present inventioncan be a useful reagent for examining caspase activity in developingtissues or cancer cells, for detecting apoptosis, or for decidingtherapeutic regimens for apoptosis-related disease.

[0073] Free text of Sequence Listing

[0074] SEQ ID NO.: 1: Synthetic peptide

[0075] SEQ ID NO.: 2: Synthetic peptide

[0076] All publications, patents and patent applications cited hereinare incorporated by reference in their entirety.

1 4 1 6 PRT Artificial Sequence Description of ArtificialSequenceSynthetic peptide 1 Cys Gln Ile Asp Val Asp 1 5 2 6 PRTArtificial Sequence Description of Artificial SequenceSynthetic peptide2 Val Ser Lys Pro Asp Cys 1 5 3 466 PRT Homo sapiens 3 Met Ser Thr ArgSer Val Ser Ser Ser Ser Tyr Arg Arg Met Phe Gly 1 5 10 15 Gly Pro GlyThr Ala Ser Arg Pro Ser Ser Ser Arg Ser Tyr Val Thr 20 25 30 Thr Ser ThrArg Thr Tyr Ser Leu Gly Ser Ala Leu Arg Pro Ser Thr 35 40 45 Ser Arg SerLeu Tyr Ala Ser Ser Pro Gly Gly Val Tyr Ala Thr Arg 50 55 60 Ser Ser AlaVal Arg Leu Arg Ser Ser Val Pro Gly Val Arg Leu Leu 65 70 75 80 Gln AspSer Val Asp Phe Ser Leu Ala Asp Ala Ile Asn Thr Glu Phe 85 90 95 Lys AsnThr Arg Thr Asn Glu Lys Val Glu Leu Gln Glu Leu Asn Asp 100 105 110 ArgPhe Ala Asn Tyr Ile Asp Lys Val Arg Phe Leu Glu Gln Gln Asn 115 120 125Lys Ile Leu Leu Ala Glu Leu Glu Gln Leu Lys Gly Gln Gly Lys Ser 130 135140 Arg Leu Gly Asp Leu Tyr Glu Glu Glu Met Arg Glu Leu Arg Arg Gln 145150 155 160 Val Asp Gln Leu Thr Asn Asp Lys Ala Arg Val Glu Val Glu ArgAsp 165 170 175 Asn Leu Ala Glu Asp Ile Met Arg Leu Arg Glu Lys Leu GlnGlu Glu 180 185 190 Met Leu Gln Arg Glu Glu Ala Glu Asn Thr Leu Gln SerPhe Arg Gln 195 200 205 Asp Val Asp Asn Ala Ser Leu Ala Arg Leu Asp LeuGlu Arg Lys Val 210 215 220 Glu Ser Leu Gln Glu Glu Ile Ala Phe Leu LysLys Leu His Glu Glu 225 230 235 240 Glu Ile Gln Glu Leu Gln Ala Gln IleGln Glu Gln His Val Gln Ile 245 250 255 Asp Val Asp Val Ser Lys Pro AspLeu Thr Ala Ala Leu Arg Asp Val 260 265 270 Arg Gln Gln Tyr Glu Ser ValAla Ala Lys Asn Leu Gln Glu Ala Glu 275 280 285 Glu Trp Tyr Lys Ser LysPhe Ala Asp Leu Ser Glu Ala Ala Asn Arg 290 295 300 Asn Asn Asp Ala LeuArg Gln Ala Lys Gln Glu Ser Thr Glu Tyr Arg 305 310 315 320 Arg Gln ValGln Ser Leu Thr Cys Glu Val Asp Ala Leu Lys Gly Thr 325 330 335 Asn GluSer Leu Glu Arg Gln Met Arg Glu Met Glu Glu Asn Phe Ala 340 345 350 ValGlu Ala Ala Asn Tyr Gln Asp Thr Ile Gly Arg Leu Gln Asp Glu 355 360 365Ile Gln Asn Met Lys Glu Glu Met Ala Arg His Leu Arg Glu Tyr Gln 370 375380 Asp Leu Leu Asn Val Lys Met Ala Leu Asp Ile Glu Ile Ala Thr Tyr 385390 395 400 Arg Lys Leu Leu Glu Gly Glu Glu Ser Arg Ile Ser Leu Pro LeuPro 405 410 415 Asn Phe Ser Ser Leu Asn Leu Arg Glu Thr Asn Leu Asp SerLeu Pro 420 425 430 Leu Val Asp Thr His Ser Lys Arg Thr Phe Leu Ile LysThr Val Glu 435 440 445 Thr Arg Asp Gly Gln Val Ile Asn Glu Thr Ser GlnHis His Asp Asp 450 455 460 Leu Glu 465 4 466 PRT Mus sp. 4 Met Ser ThrArg Ser Val Ser Ser Ser Ser Tyr Arg Arg Met Phe Gly 1 5 10 15 Gly SerGly Thr Ser Ser Arg Pro Ser Ser Asn Arg Ser Tyr Val Thr 20 25 30 Thr SerThr Arg Thr Tyr Ser Leu Gly Ser Ala Leu Arg Pro Ser Thr 35 40 45 Ser ArgSer Leu Tyr Ser Ser Ser Pro Gly Gly Ala Tyr Val Thr Arg 50 55 60 Ser SerAla Val Arg Leu Arg Ser Ser Val Pro Gly Val Arg Leu Leu 65 70 75 80 GlnAsp Ser Val Asp Phe Ser Leu Ala Asp Ala Ile Asn Thr Glu Phe 85 90 95 LysAsn Thr Arg Thr Asn Glu Lys Val Glu Leu Gln Glu Leu Asn Asp 100 105 110Arg Phe Ala Asn Tyr Ile Asp Lys Val Arg Phe Leu Glu Gln Gln Asn 115 120125 Lys Ile Leu Leu Ala Glu Leu Glu Gln Leu Lys Gly Gln Gly Lys Ser 130135 140 Arg Leu Gly Asp Leu Tyr Glu Glu Glu Met Arg Glu Leu Arg Arg Gln145 150 155 160 Val Asp Gln Leu Thr Asn Asp Lys Ala Arg Val Glu Val GluArg Asp 165 170 175 Asn Leu Ala Glu Asp Ile Met Arg Leu Arg Glu Lys LeuGln Glu Glu 180 185 190 Met Leu Gln Arg Glu Glu Ala Glu Ser Thr Leu GlnSer Phe Arg Gln 195 200 205 Asp Val Asp Asn Ala Ser Leu Ala Arg Leu AspLeu Glu Arg Lys Val 210 215 220 Glu Ser Leu Gln Glu Glu Ile Ala Phe LeuLys Lys Leu His Asp Glu 225 230 235 240 Glu Ile Gln Glu Leu Gln Ala GlnIle Gln Glu Gln His Val Gln Ile 245 250 255 Asp Val Asp Val Ser Lys ProAsp Leu Thr Ala Ala Leu Arg Asp Val 260 265 270 Arg Gln Gln Tyr Glu SerVal Ala Ala Lys Asn Leu Gln Glu Ala Glu 275 280 285 Glu Trp Tyr Lys SerLys Phe Ala Asp Leu Ser Glu Ala Ala Asn Arg 290 295 300 Asn Asn Asp AlaLeu Arg Gln Ala Lys Gln Glu Ser Asn Glu Tyr Arg 305 310 315 320 Arg GlnVal Gln Ser Leu Thr Cys Glu Val Asp Ala Leu Lys Gly Thr 325 330 335 AsnGlu Ser Leu Glu Arg Gln Met Arg Glu Met Glu Glu Asn Phe Ala 340 345 350Leu Glu Ala Ala Asn Tyr Gln Asp Thr Ile Gly Arg Leu Gln Asp Glu 355 360365 Ile Gln Asn Met Lys Glu Glu Met Ala Arg His Leu Arg Glu Tyr Gln 370375 380 Asp Leu Leu Asn Val Lys Met Ala Leu Asp Ile Glu Ile Ala Thr Tyr385 390 395 400 Arg Lys Leu Leu Glu Gly Glu Glu Ser Arg Ile Ser Leu ProLeu Pro 405 410 415 Thr Phe Ser Ser Leu Asn Leu Arg Glu Thr Asn Leu GluSer Leu Pro 420 425 430 Leu Val Asp Thr His Ser Lys Arg Thr Leu Leu IleLys Thr Val Glu 435 440 445 Thr Arg Asp Gly Gln Val Ile Asn Glu Thr SerGln His His Asp Asp 450 455 460 Leu Glu 465

What is claimed is:
 1. An antibody reacting with a cleavage product ofvimentin but not reacting with intact vimentin.
 2. The antibody of claim1 wherein the cleavage product of vimentin is obtained by action of acaspase.
 3. The antibody of claim 2 wherein the caspase is caspase-8. 4.The antibody of any one of claims 1 to 3 wherein the antibody is apolyclonal antibody or a monoclonal antibody.
 5. A method for detectingcaspase activity comprising allowing the antibody of any one of claims 1to 4 to react with the cleavage product of vimentin and detecting theresulting reaction product.
 6. A method for detecting apoptosiscomprising allowing the antibody of any one of claims 1 to 4 to reactwith the cleavage product of vimentin and detecting the resultingreaction product.
 7. A reagent for detecting apoptosis containing theantibody of any one of claims 1 to 4.